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proceeded to select novel NAG variants with enhanced activity. A
further top 0.2% of cells were sorted in the second round. After
each round, half of the sorted samples were plated while the
other half were grown for another round of sorting. Individual
colonies were randomly sequenced colonies from each round of
sorting (representative results in Table S3, ESI†).
The NAG enzyme is grouped within the GH109 family of glycosi-
dases. Its structure has been established, revealing residues Tyr-307,
Tyr-225, His-228 and Glu-149 are involved with substrate binding in
the enzyme active site.13 Based on the sequences and structural
positions of mutation sequences, we selected five Round-2 colonies
with mutations near the active site for follow-up (Table S3, ESI†).
However, we could only successfully purify the enzyme from one of the
colonies and hence quantified its activity profiles carefully. The yields
from the other variants were extremely low, when purification was
attempted under native conditions. The purified variant carried two
non-synonymous mutations (H104L and S347R) (Table S2 and Fig. S3,
ESI†). Both these mutations are located within 20 Å of the enzyme
active site (Fig. 4C). This variant NAG was found to exhibit a lower KM
and close to two times higher kcat/KM ratio than the wildtype NAG
(Fig. 4). This correlated well with expectations from the FACS experi-
ments. No further variants with enhanced activities were identified.
In conclusion, we evolved a sub-optimal enzyme to a more
active form using the smart qABP strategy developed herein.
Compared to traditional fluorogenic probes, quinone methide
qABPs do not diffuse out of cells readily, so continued measure-
ments of the fluorescence signals will be possible. As far as we
know, this is the first example of using such smart quinone
methide-based probes for evolving enzyme activity. The novel
NAG variant identified will be tested on human RBCs to
evaluate its efficacy. We anticipate that this strategy may be
applied more broadly in enzyme engineering and evolution.
The authors acknowledge funding support from DSO
National Laboratories, Singapore (Grant no. 20090211). Ms
Tan Pei Xin developed the NAG constructs used in this study.
Fig. 3 FACS sorting results. (A) Wildtype NAG expressing cells sorted with and
without Probe A (red and blue histograms respectively). (B) Mutant library sorted
with and without probe (red and blue histograms respectively). (C) The bottom
0.5% and the top 0.2% library populations were re-grown and sorted, generat-
ing the distinct green and orange histograms respectively.
population of positively fluorescent cells was obtained consistently. A
significant proportion of the cell population generated fluorescence
readouts above 100 RFUs, with greater than 95% of the cell popula-
tion producing consistent readouts greater than 10 RFUs. By incu-
bating the cell population for extended period of times on ice (to
limit cell division), we found that the cells produced nearly identical
sorting profiles, and the probe was successfully retained in the cells
for at least several hours without significant loss of fluorescence.
Thereafter, we tested the library of NAG variants with Probe A.
We found that a large fraction of electroporated mutant library
cells incubated with Probe A emitted fluorescence (Fig. 3B, red
curve), when compared to the group that contained
Novablue(DE3) cells without Probe A (Fig. 3B, blue curve). In
order to test that we could select desired populations of active
variants, we proceeded to sort out the top 0.2% of the electro-
porated mutant library. We also sorted the bottom 0.5% of the
library. These two populations were then re-grown in culture and
studied using FACS (Fig. 3B, orange and green curves respec-
tively). The results showed that there was a marked difference in
the activity of the sorted cell populations, indicating that higher-
activity variants could be enriched using the strategy. We thus
Notes and references
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Fig. 4 (A) Kinetic activities of the control and the variant NAG enzymes (H104L,
S347R) were plotted on a Michaelis–Menten plot (averaged from triplicates). (B)
KM and kcat values of the NAG wildtype and variant were calculated by non-linear
regression, least squares fitting, ꢁ95% confidence intervals. (C) The wildtype NAG
enzyme is shown on the left. The variant NAG enzyme (with protein ribbon in red)
has mutated amino acids H104L, S347R in black. The active site residues are shown
in blue. Figures are adapted from PDB entry 2IXA13 and rendered in Pymol.
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c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 7237--7239 7239